Method for extracting usnic acid



Patented June 26, 1951 UNITED STATES PATENT OFFICE N Drawing.Application September 9, 1947, Serial No. 773,067

3 Claims.

(Granted under the act of March 3, 1883, as amended April 30, 1928; 3700. G. 757) The invention described herein may be manufactured and usedby or for the Government of the United States for governmental purposeswithout the payment to me of any royalty thereon in accordance with theprovisions of the act of April 30, 1928 (Ch. 460, 45 Stat. L. 467).

This invention relates to therapeutic compositions and methods ofadministering them. More particularly this invention relates to anantibiotic crystalline substance derived from the lichen Ramalz'nareticulum and means and methods for administering this antibiotic toanimals.

This application is a continuation-in-part of my application Serial No.7 38,503, filed March 31, 1947, now abandoned, entitled TherapeuticComposition and Method.

Since the discovery of the tubercule bacillus repeated and persistentefforts have been made to find a drugor antibiotic that would beeffective in the cure of tuberculosis. Tremendous effort over a greatlength of time has been expended to find a lethal agent to defeat a germthat has consistently resisted every attempt against its predatoryexistence. Over the years the hopes of the ill have been lifted by suchattempts at treatment as tuberculin injections, gold therapy, theapplications of sulpha drugs, and various vaccines. In every instancethe high hopes were dashed by failure.

It is an object of this invention to provide a new antibiotic.

It is a further object of this invention to provide a new compound toxicto tubercule bacilli in vitro.

It is a further object of this invention to provide a new compound toxicto tubercule bacilli in vivo.

It is a further object to provide a method of extracting an antibioticfrom the lichen Rama- Zina reticulata.

It is a further object to provide a new com pound toxic to the tuberculebacillus and capable of being absorbed by an animal system.

It is a further object to provide a method of and means foradministering therapeutics to animals.

The foregoing and other objects hereinafter apparent are accomplished inaccordance with this invention wherein a therapeutic'composition iscaused to be assimilated in an animal circulatory system by beingdissolved in oil and combined with a dispersing, wetting and/oremulsifying agent and injected in said animal, and wherein there isprovided an antibiotic substance and a method of extracting thisantibiotic substance from the lichen Ramalina reticulata.

Ramalina reticulata (Noedh.) Kremph, sometimes called California Spanishmoss, is a lichen of the family Usneaceae which grows as an epiphytealong the West coast of North America from California to Alaska. Theplant has no integument but does contain in the interstices betweenhyphae and alga cells a carbohydrate substance which is veryhygroscopic, so that under foggy conditions it is soft, friable, andsaturated with water. The eastern and the California Spanish moss arenot in any way related. The former is a seed plant of the familyBromeliaceae and the latter is a lichen.

Method of attraction The antibiotic may be extracted from the lichenRamalz'na reticulum by any of the following methods:

Method 1.--The lichen is extracted by boiling for four hours withacetone (2 parts) and alcohol (1 part). The extract is filtered andafter standing at room temperature with slow evaporation for a week acopious green preciptate appears which is filtered off. The precipitateis dissolved in boiling acetone and filtered while hot. On coolingyellow needle-like crystals appear.- The green mother liquor is decantedand the crystals washed with alcohol and acetone. The crystals are againdissolved (the solution is now yellow) recrystallized and washed. Thisprocess of dissolving, recrystallization and washing is repeated twomore times. With slow crystallizaton crystals as long as one inch can beobtained.

Method 2.-The cleaned lichen is packed in 6,-gallon earthenware crooksand covered with acetone (2 parts) and alcohol (lpart). Approximately 10pounds of lichen to pounds of acetone alcohol are employed. Afterstanding ap proximately 12 hours the yellow green solution is decanted,filtered, and poured into enamelware pans, the pans are put outdoors andprotected against direct sunlight. In a brisk breeze evaporationproceeds rapidly and in a few hours a copious green precipitate appearsand is filtered off. The red brown mother liquor is evaporated furtheruntil only an amorphous tan precipitate is produced. The greenprecipitate is dissolved in boiling acetone and filtered rapidly whilehot. The filtrate is then concentrated by boiling to about one-tenth itsorignal volume. On cooling, crystallization occurs rapidly. The greenmother liquor is then decanted and the yellow crystals are washed with,cold acetone. The process of.

3 dissolving, recrystallizing and washing is repeated two more times.

Method 3.Extraction with cold acetone is carried out as described inmethod 2. The mother liquor in this case is yellow. The separation ofcrystals from amorphous material was carried out as in method 2.

Method 4.58 grams of lichen are extracted with 600 cc. of chloroform atroom temperature for about 21 hours. The solvents are removed byevaporation and the sediments dissolved in hot acetone. On reducting thevolume of the acetone. to 1/4-V3 of the original volume, crystallinepreciptates are obtained which are dissolved, recrystallized and washedtwo more times.

The yield by methods 2 and 3 is approximately 8 gm. of purifiedcrystalline material per 10 pounds of lichen. The yield by method 4 isapproximately 16 gm. of purified crystalline material per 10 pounds oflichen, the yield by chloroform extraction being approximately twice aslarge as that obtained by acetone extraction, where equal volumes ofeach solvent per unit weight of lichen are compared.

Properties of the crystalline material The crystalline materialextracted from the lichen Ramalina reticulum by any of the foregoingmethods is readily soluble in hot acetone, ethyl alcohol, propyleneglycol, ethyl ether, chloroform, carbon tetrachloride; poorly soluble inhot petroleum ether, cold alcohol, propylene glycol; and moderatelysoluble in cold acetone. The material is insoluble in water and in HCl.

Solubility studies have shown that the pure crystalline material isabout 10 times more soluble in chloroform than in acetone, thusindicating that it may be possible to extract the crystals from thelichen with a volume r" chloroform roughly /20 the volume of the acetonewhich would have to be used for the same process.

The melting point of the crystalline material is l91-l92 C. when heatedat an increment of 02 per minute, after first being brought rapidly to160 C. The melting point is 192-194" C. when heated at a uniformincrement of 0.5-1.0 per minute. The melt is brown. The crystalsobtained when the melt cools are yellow-brown. The crystals melt readilyin camphor. However, when the mixture is again heated there appears tobe progressive decomposition with no definite melting point.

The substance is acid and has a neutralization equivalent of 298-310, asmeasured by titration in acetone.

0n analysis, no ash, nitrogen, or halide is found. The percentagecomposition of the crystalline material obtained with hot acetoneextraction is (a) C62.75, H--l.63, (b) C62.'75, H4.69. Analysis of thematerial extracted with cold acetone-alcohol gives a percentagecomposition of (a) 6-63.05, H--4.49, (b) C-63.00, Iii-4.64. The materialcontains no methoxyl groups. The results of analysis indicate one groupper molecule titratable as an acid. Original analysis was done with amethoxyl derivative of the extract because of doubt concerning thepurity of the parent compound. Analysis of the methoxyl derivative gaveanalytical data which fitted the empirical formula ClGHMOS. Laterinvestigation indicated that the extracted compound was homogeneous andanalysis was carried out on the parent compound.

The parent compound extracted from the lichen ,Ramalz'na reticulata fromthe accumulated analy- 4 tical data, homogeneity studies, ultra violetand infra red spectroscopy, optical rotation and X-ray analysis, isstrongly suggested to be d-usnic acid, with an empirical formulaClSHlSO'].

Antibacterial properties in vitro The growth of the bacteriaPneumococcus, Streptococcus and some or the Staphyiococci are inhibitedby 50 gamma per cc. or less. Experiments designed to define more closelythe minimal effect of concentration in strains of Pneumococcus andStreptococcus showed complete sterilization with concentrations of 10 to20 gamma per co. in the former while in the latter the variation betweenstrains was much greater, i. e., from 10 to over 50 gamma per cc. Thehuman strains of tubercle bacilli showed complete inhibition byconcentrations of l to 50,000 and noticeable inhibition atconcentration. as low as 2 to 2,000,000 with the exception of the Wallerstrain which required a concentration of l to 20,000 i or complete, and1 to 200,000 for partial inhibition.

Pro erties in vice Saline suspensions of the crystalline material do notprovide an adequate means for dispersing the antibacterial agent in theanimal body. Solutions in oil alone are also unsuitable since a gooddeal of the oil remains in situ after injection although some oil may beincorporated into the fat cells. The crystalline substance may bedissolved in oil by being first dissolved in acetone which is combinedwith the oil, the acetone then being distilled off. By dissolving thecrystalline material in sesame oil and combining the oil with adispersing, emulsifying or wetting agent such as Tween (polyoxyallryleneether of sorbitan monooleate) in suitable proportions is was possible tohave the oil taken up into the animal circulatory system with no obviouslocal or systemic injury. In mice (25 gm.) one-half to one mg. in 0.15cc. oil containing 10 percent Tween 80 could be taken up withoutappreciable damage while 0.2 mg. in a single dose was lethal. 30 mg. ofthe crystalline material given twice daily to 250-350 gm. guinea pigswas lethal, while 20 mg. given once daily was not. In guinea pigs(350-400 gm.) a single 1 cc. dose of Tween 80 and oil having greateramounts of Tween than 1 part to 9 parts of oil produced swelling of thefascia. Repeated daily injections in guinea pigs (350-400 gm.) of 1 cc.of a mixture of 1 part Tween 80 to 9 parts oil produced swelling of thefascia after three days, but a mixture of 0.1 cc. of 20% Tween 80 insaline (.85% sodium chloride in distilled water) plus 0.9 cc. of oilgiven in 0.5 cc. daily injections did not produce swelling. 20 mg. ofthe crystalline material given guinea pigs (250-320) in a 1 cc. emulsionby a single injection is not completely absorbed after four days,whereas, 10 mg. in the same volume of emulsion is completely absorbed intwo days. 0.1 cc. of a 20 percent solution of Tween 80 in saline addedto .9 cc. oil produces a fine emulsion stable for about l8 hours. Thecrystals are dissolved in oil and the solution emulsified in the saline.When emulsified some of the crystalline material precipitates out but iseasily resuspended. At room temperature the mixture ofcrystals-Tween-oil-saline appears as an emulsion with some of thecrystalline material dispersed in it as a solid. At the temperature ofthe body (37 C.) however, the crystalline substance is completelydissolved in this mixture.

In a test lasting thirty-two days a number of guinea pigs wereintraperitoneally inoculated with .01 mg. of tubercle bacilli. The groupof guinea pigs receiving daily subcutaneous injections of between and 20mg. of the antibiotic crystalline material in oil combined with Tween 80and saline for 21 days days lost less than half as much weight as theinfected guinea pigs not receiving the antibiotic treatment.

Reference is made to Public Health Reports" for January 3, 1947, volume62, No. 1, printed by the U. S. Public Health Service, pages 3-19, for amore complete disclosure of the tests made with the crystallineantibacterial substance derived from the lichen Ramalina recticulata inretarding the progress of human strains of the tubercle bacilli inanimals.

It will be appreciated that the invention is susceptible of variouschanges without depart ing from the spirit and scope thereof, as setforth in the appended claims.

What is claimed is:

1. A process for isolating usnic acid from the lichen Ramalz'nareticulata which comprises treating said lichen with a solvent selectedfrom the group consisting of chloroform and acetone, filtering theextract to remove the insoluble elements of said lichen therefrom,maintaining said extract filtrate at substantially room temperature fora time suflicient to allow a precipitate to form therein, dissolvingsaid precipitate in a solution of hot acetone, and then cooling the thusformed solution whereby crystals of usnic acid are formed.

2. A process for isolating usnic acid from the lichen Ramalinarecticulata which comprises treating said lichen with acetone, filteringthe extract to remove the insoluble elements of said lichen therefrom,maintaining said extract 11- trate at substantially room temperature fora time suflicient to allow a precipitate to form therein, dissolvingsaidprecipitate in a solution of hot acetone, and then cooling the thusformed solution whereby crystals of usnic acid are formed. *1

3. A process for isolating usnic acid from the lichen Ramalinarettcitld'ta which comprises treating said lichen with cholorform,filtering the extract to removetli'e insoluble elements of said lichentherefrom, maintaining said extract filtrate at substantially roomtemperature for a time sufficient to allow a precipitate to formtherein, dissolving said precipitate in a solution of hot acetone, andthen cooling the thus formed solution whereby crystals of usnic acid areformed.

ALFRED G. MARSHAK.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 1,796,070 Waterhouse Mar. 10,1931 2,017,596 Hofiman Oct. 15,1935 2,059,811 Sauer Nov. 3, -19362,170,872 Peebles Aug. 29, 1939

1. A PROCESS FOR ISOLATING USNIC ACID FROM THE LICHEN RAMALINARETICULATA WHICH COMPRISES TREATING SAID LICHEN WITH A SOLVENT SELECTEDFROM THE GROUP CONSISTING OF CHLOROFORM AND ACETONE, FILTERING THEEXTRACT TO REMOVE THE INSOLUBLE ELEMENTS OF SAID LICHEN THEREFROM,MAINTAINING SAID EXTRACT FILTRATE AT SUBSTANTIALLY ROOM TEMPERATURE FORA TIME SUFFICIENT TO ALLOW A PRECIPITATE TO FORM THEREIN, DISSOLVINGSAID PRECIPITATE IN A SOLUTION OF HOG ACETONE, AND THEN COOLING THE THUSFORMED SOLUTION WHEREBY CRYSTALS OF USNIC ACID ARE FORMED.